Library

Notes: Abstract: Endogenous material present in heat-denatured extracts of rat brain that inhibited the binding of [3H]-isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-5-methoxycarbonyl-2,6-dimethyl-3-pyridinecarboxylate ([3H]-PN200-110) to calcium channels in brain membranes was purified. Spectrophotometric analysis of material purified by strong anion-exchange and reverse-phase chromatography showed an absorption maximum at 266 nm at pH 7.0 that shifted to 245 nm at pH 2.0. This pH-dependent spectral shift was indistinguishable from that of ascorbic acid. Samples of the purified extract contained ascorbic acid; however, the inhibition of binding by purified material was always greater than the inhibition seen with equivalent concentrations of ascorbate, implying the presence of additional inhibitory factors. Attempts to detect and identify such inhibitory substances by chromatography showed that inhibitory activity was coincident with the presence of ascorbate, and the inhibitory activity of purified material was abolished after treatment with ascorbic acid oxidase. Iron enhanced the inhibition produced by ascorbate, and chemical analysis of purified preparations revealed the presence of iron. Studies comparing the potency of the purified material with that of a mixture of ascorbate plus iron showed that the content of ascorbate and iron in the purified brain extract is sufficient to explain the observed inhibition of binding of [3H]PN200-110.

Notes: Abstract: Most antibodies known to interact with β-adren-ergic receptors do not exhibit subtype selectivity, nor do they provide quantitative immunoprecipitation. A monoclonal antibody, G27.1, raised against a synthetic peptide corresponding to the C-terminus of the β2-adrenergic receptor of hamster, is selective for the β2 subtype. G27.1 provides nearly quantitative immunoprecipitation of the β2-acjrenergic receptor from hamster lung that has been photoaffinity-labeled and solubilized with sodium dodecyl sulfate. Immunoprecipitation is completely blocked by nanomolar concentrations of the immunizing peptide. This antibody interacts with β2-adrenergic receptors from three rodent species, but not with those from humans. When C6 glioma cells, which contain both β1 - and β2-adrenergic receptors, are photoaffinity-labeled in the absence or presence of subtype-selective antagonists, subtype-selective photoaffinity-labeling results. G27.1 can immunoprecipitate β-, but not β1-, adrenergic receptors from these cells. Similar results were obtained following subtype-selective photoaffinity-labeling of membranes from rat cerebellum and cerebral cortex. The β-adrenergic receptors from C6 glioma cells and rat cerebral cortex exist as a mixture of two molecular weight species. These species differ in glycos-ylation, as shown by endoglycosidase F digestion of crude and immunoprecipitated receptors.

Notes: Spermine and spermidine enhance the binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]cyclohepten-5,10-imine ([3H]MK-801) to N-methyl-D-aspartate (NMDA) receptors in membranes prepared from rat brain. These polyamines also enhance binding of [3H]MK-801 to NMDA receptors that have been solubilized with deoxycholate. Other polyamines selectively antagonize this effect, a finding indicating that the polyamine recognition site retains pharmacological and structural specificity after solubilization. In the presence of spermidine, an increase in the affinity of the solubilized NMDA receptor for [3H]MK-801 is observed. However, the rates of both association and dissociation of [3H]MK-801 binding to solubilized NMDA receptors are accelerated when assays are carried out in the presence of spermidine. When kinetic data are transformed, pseudo-first-order association and first-order dissociation plots are nonlinear in the presence of spermidine, an observation indicating a complex binding mechanism. Effects of spermidine on solubilized NMDA receptors are similar to effects previously described in studies of membrane-bound receptors. The data indicate that polyamines interact with a specific recognition site that remains associated with other components of the NMDA receptor complex after detergent solubilization.

Notes: Abstract: 125I-SCH 23982, an antagonist with high affinity and selectivity for the D-l subtype of dopamine receptors, has recently been synthesized. Densities of D-l receptors in rat brain obtained from autoradiographic studies using this iodinated ligand are 5- to 10-fold less than densities reported with tritiated analogues such as [3H]SCH 23390. A direct comparison of these two ligands using striatal ho-mogenates confirmed this discrepancy. One explanation for this difference is that 125I-SCH 23982 labels a subset of the sites labeled by [3H]SCH 23390. However, the distributions of sites labeled by the ligands in autoradiograms of horizontal sections of rat brain were virtually identical. Furthermore, 127I-SCH 23982 displaced 100% of the specifically bound [3H]SCH 23390 in striatal homogenates with a Hill coefficient of approximately 1. These results are not consistent with the existence of a subset of receptors recognized by 125I-SCH 23982 and suggest that both ligands label the same population of receptors. An alternative explanation for the discrepancy in 5max values is that an unlabeled inhibitor is present in commercial preparations of 125I-SCH 23982. When all of the solvent (including any volatile inhibitors) was removed from commercial preparations of 125I-SCH 23982 prior to use in radioligand binding experiments, the discrepancy in Bmax values was eliminated.

Notes: Abstract: The role of the a subunit of the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase (Gsα) in the down-regulation of β-Adrenergic receptors by pindolol was studied in S49 cyc− cells (normally Gsα-deficient) transfected to express functional recombinant rat Gsα. An inducible cell line (S49 GsαIND) was derived from S49 cyc− cells transfected with a vector containing the full-length coding sequence of Gsα under the inducible control of the mouse mammary tumor virus long-terminal repeat promoter. Gsα was not detectable in S49 GsαIND cells by immunoblot or by ADP-ribosylation in the presence of cholera toxin and [α-32P]NAD. When cells were grown in 100 nM dexamethasone, isoproterenol-stimulated cyclic AMP accumulation increased within 3 h. After 15 h, Gsα was present at a level 40–50% of that found in S49 wild-type (WT) cells as measured either by immunoblot analysis or by [α-32P]ADP-ribosylation. Membranes prepared from GsαIND cells grown in the presence of dexamethasone bound agonist with high affinity, and this binding was sensitive to guanine nucleotides. A second vector, DzbGsα+, contained the coding sequence of Gsα under the constitutive regulatory control of the SV40 early promoter. This vector was introduced into cyc− cells, and the resulting cells, S49 GsαCST cells, expressed Gsα at a level comparable to that found in S49 WT cells as measured by immunoblot analysis. Isoproterenol-stimulated cyclic AMP accumulation in S49 GsαCST cells was at least as great as in S49 WT cells. When cells were grown in the presence of dexamethasone, exposure to 50 nM pindolol for 12 h down-regulated the density of 0-adrenergic receptors in S49 WT cells to 60% of that in cells grown in the absence of pindolol, but pindolol had no effect on the density of receptors on cyc− or Gsα IND cells. When Gsα CST cells were exposed to 50 nM pindolol for 12 h, the density ofβ-Adrenergic receptors was down-regulated by the same amount as in S49 WT cells. These results suggest that Gsα is necessary to restore the ability of pindolol to down-regulate β-adrenergic receptors in S49 cyc~ cells and that the protein must be expressed at a level comparable to that found in S49 WT cells.

Notes: Abstract: A diverse panel of monoclonal antibodies was obtained from BALB/c mice immunized with two haptens structurally related to spiroperidol (SPD). Bromoacetyl derivatives of aminospiroperidol (NH2SPD) and N-amino-phenethylspiroperidol (NAPS) were synthesized to couple the haptens covalently to a protein carrier for immunization, thereby maintaining the butyrophenone portion of the immunogen. Hybridomas were selected based on their ability to secrete antibody that binds [3H]SPD with high affinity. Equilibrium dissociation constants for these antibodies ranged from 0.2 to 〉 100 nM. The antigen binding sites of the anti-NH2SPD and anti-NAPS antibodies were characterized in studies of the inhibition of the binding of [3H]-SPD by a series of ligands that are either (a) structurally related to SPD or (b) structurally unrelated to the butyrophenones but known to be selective antagonists of the D2 subtype of dopamine receptor. Based on the patterns of inhibition of the binding of [3H]SPD by these compounds, 12 classes of antibody combining sites were identified. Most of these antibodies bound butyrophenones with high affinity. One anti-NH2SPD and four anti-NAPS antibodies also bound domperidone, a nonbutyrophenone that has a high affinity for D2 receptors. None of the antibodies bound clebopride or sulpiride, D2-selective antagonists of the benzamide class, or the agonist dopamine.

Notes: Abstract: Developmental changes in the levels of N-methyl-d-aspartate (NMDA) receptor subunit mRNAs were identified in rat brain using solution hybridization/RNase protection assays. Pronounced increases in the levels of mRNAs encoding NR1 and NR2A were seen in the cerebral cortex, hippocampus, and cerebellum between postnatal days 7 and 20. In cortex and hippocampus, the expression of NR2B mRNA was high in neonatal rats and remained relatively constant over time. In contrast, in cerebellum, the level of NR2B mRNA was highest at postnatal day 1 and declined to undetectable levels by postnatal day 28. NR2C mRNA was not detectable in cerebellum before postnatal day 11, after which it increased to reach adult levels by postnatal day 28. In cortex, the expression of NR2A and NR2B mRNAs corresponds to the previously described developmental profile of NMDA receptor subtypes having low and high affinities for ifenprodil, i.e., a delayed expression of NR2A correlating with the late expression of low-affinity ifenprodil sites. In cortex and hippocampus, the predominant splice variants of NR1 were those without the 5′ insert and with or without both 3′ inserts. In cerebellum, however, the major NR1 variants were those containing the 5′ insert and lacking both 3′ inserts. The results show that the expression of NR1 splice variants and NR2 subunits is differentially regulated in various brain regions during development. Changes in subunit expression are likely to underlie some of the changes in the functional and pharmacological properties of NMDA receptors that occur during development.

Notes: Abstract: Portions of the cDNA encoding the third intracellular loop (i3 loop) of the long and short isoforms of the rat D2 dopamine receptor were subcloned into the vector pNMHUBpoly and expressed in Escherichia coli as fusion proteins. The fusion proteins were gel-purified and used to immunize rabbits for the production of polyclonal anti-receptor antisera. The anti-fusion protein antisera recognized synthetic peptides corresponding to segments of the i3 loops of D2 dopamine receptors in a solid-phase radioimmunoassay. Antisera were tested in an immunoprecipitation assay using the reversible D2 antagonist [125I]NCQ 298 and digitonin-solubilized extracts of canine and rat caudate. [125I]-NCQ 298 bound reversibly and with high affinity (KD= 0.14 nM) to receptors in solubilized extracts enriched by chromatography on heparin-agarose. The anti-UBI-D2i3L and anti-UBI-D2i3s antisera were able to immunoprecipitate quantitatively D2 dopamine receptors labeled with [125I]NCQ 298 from solubilized rat caudate. The antibodies were tested for their ability to affect the coupling of D2 dopamine receptors to GTP-binding proteins in digitonin-solubilized rat caudate. Both anti-UBI-D2i3L and anti-UBI-D2i3s antisera were able to inhibit the high-affinity binding of the agonist N-propylnorapomorphine to digitonin-solubilized rat caudate. These findings indicate that the i3 loop of the D2 dopamine receptor is an important determinant for coupling of the G protein.